Enzymatic methylation of cytosine bases at carbon 5 resulting in 5-methyl-cytosine is a DNA modification found in prokaryotes as well as eukaryotes. In eukaryotic cells it is well known as a fundamental epigenetic mechanism resulting in gene silencing. Methylation occurs specifically at CpG dinucleotides which are frequently found within promoter regions. Outside promotors, CpG sites are rare. Methylated promotors are silent i.e. transcriptionally inactive so that the gene is not expressed. Methylated DNA recruits methyl-DNA binding proteins which form a platform for other factors like HDACs which modify and condense the chromatin.
Gene silencing by promoter methylation has been reported not only for autologous genes but also for recombinant gene constructs (Escher, G., et al., J. Lipid Res. 46 (2005) 356-365; Brooks, A. R., et al., J. Gen. Med. 6 (2004) 395-404). Bisulfite treatment of DNA is a method suitable to discriminate between methylated and non-methylated CpG sites. Under the conditions used, cytosine but not 5-methyl-cytosine is deaminated at C4 and thereby converted to uracil. Complementary DNA strands are thereby converted into two strands, A and B, which are no longer complementary.
Barnes, L. M., et al. (Biotechnol. Bioeng. 96 (2006) 337-348) report that the LC/HC mRNA level is for NSO cell a marker for long-term stability. Chusainow, J., et al. (Biotechnol. Bioeng. 102 (2009) 1182-1196) and Jiang, Z., et al. (Biotechnol. Prog. 22 (2006) 313-318) report that higher LC/HC mRNA levels for MTX treated cells. Lee, C. J., et al. (Biotechnol. Bioeng. 102 (2008) 1107-1118) report the screening of cells based on the HC mRNA level.
Escher et al. (see above) report CMV promotor silencing in transiently transfected macrophages. CMV promoter silencing in vivo with adenoviruses is reported by Brooks et al. (see above).
VEZF1 elements mediate protection from DNA methylation is reported by Dickson, J., et al. (PLoS Genetics 6 (2001) e1000804). Mielke, C., et al. (Gene 254 (2000) 1-8) report stabilized, long-term expression of heterodimeric proteins from tricistronic mRNA. Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent adenoviral vectors is reported by Everett, R. S., et al. (J. Virol. 325 (2004) 96-105). Proesch, S., et al. (Biol. Chem. Hoppe-Seyler 377 (1996) 195-201) report inactivation of the very strong HCMV immediate early promoter by DNA CPG methylation in vitro. PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment is reported by Kristensen, L. S. and Hansen, L. L. (Clin. Chem. 55 (2009) 1471-1483). Recillas-Targa, F., et al. (Proc. Natl. Acad. Sci. USA 99 (2002) 6883-6888) report that position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities.